Phosphorylcholine is an important bioactive adduct to the teichoic acid (TA) and lipoteichoic acid (LTA) of the surface of Streptococcus pneumoniae. We have identified and characterized a genetic locus lic that is required for phosphorylcholine metabolism in S. pneumoniae. The pneumococcal lic locus consists of eight genes, licA, licB, licC and licD1, licD2 and three additional open reading frames. Pneumococcal licA, licB, licC, licD1 and licD2 have significant sequence similarity to licA, licB, licC and licD of Haemophilus influenzae. Mutation of licD2 led to decreased [3H]-choline uptake, aberrant migration of LTA chains in SDS-PAGE gels, loss of several surface proteins, and a phase-locked hypertransparent colony phenotype. Moreover, the licD2- mutant falled to undergo lysis after treatment with penicillin at high cell density and showed decreased transformation competence. Finally, the licD2- mutant demonstrated decreased adherence to the human type II alveolar cells, reduced nasopharyngeal colonization in infant rats, as well as significantly impaired virulence upon intraperitoneal challenge of CF1 mice. Identification of the lic genes in the pneumococcus will facilitate further characterization of the role of surface choline in microbial physiology and pathogenesis.